Targeting Ewing sarcoma with activated and GD2-specific chimeric antigen receptor-engineered human NK cells induces upregulation of immune-inhibitory HLA-G

Abstract

Activated and expanded natural killer (NK) cells have substantial cytotoxicity against many tumor cells, but their efficacy to eliminate solid cancers is limited. Here, we used chimeric antigen receptors (CARs) to enhance the activity of NK cells against Ewing sarcomas (EwS) in a tumor antigen-specific manner. Expression of CARs directed against the ganglioside antigen G in activated NK cells increased their responses to G+ allogeneic EwS cells and overcame resistance of individual cell lines to NK cell lysis. Second-generation CARs with 4-1BB and 2B4 co-stimulatory signaling and third-generation CARs combining both co-stimulatory domains were all equally effective. By contrast, adoptive transfer of G-specific CAR gene-modified NK cells both by intratumoral and intraperitoneal delivery failed to eliminate G-expressing EwS xenografts. Histopathology review revealed upregulation of the immunosuppressive ligand HLA-G in tumor autopsies from mice treated with NK cells compared to untreated control mice. Supporting the relevance of this finding, co-incubation of NK cells with allogeneic EwS cells induced upregulation of the HLA-G receptor CD85j, and HLA-G1 expressed by EwS cells suppressed the activity of NK cells from three of five allogeneic donors against the tumor cells. We conclude that HLA-G is a candidate immune checkpoint in EwS where it can contribute to resistance to NK cell therapy. HLA-G deserves evaluation as a potential target for more effective immunotherapeutic combination regimens in this and other cancers.

CAR-Expressing Natural Killer Cells for Cancer Retargeting

“…In preclinical studies, it could be shown that disialoganglioside- (GD 2 ; expressed on tumours of neuroectodermal origin [68]) positive Ewing sarcoma (ES) cells could be used for the engineering of a second- and third-generation anti-GD 2 -CAR. This led to an improvement of the NK cell-driven cytotoxicity against several cancer cell lines in vitro and resulted in effective counteraction against ES tumour resistance [69, 70]. However, adoptive transfer of anti-GD 2 -CAR-NK cells in GD 2 -expressing ES xenograft models could not confirm previous in vitro experiments [70].…”

“…Furthermore, the diminished anti-tumour response of those CAR-modified NK cells was connected to increased expression levels of immunosuppressive human leukocyte antigen G (HLA-G) on ES cells, thereby mediating tumour immune escape [71]. The authors concluded that HLA-G represents a potential checkpoint, which would have to be inhibited before initiating therapies based on CAR-NK cells [70]. …”

“…This led to an improvement of the NK cell-driven cytotoxicity against several cancer cell lines in vitro and resulted in effective counteraction against ES tumour resistance [69, 70]. However, adoptive transfer of anti-GD 2 -CAR-NK cells in GD 2 -expressing ES xenograft models could not confirm previous in vitro experiments [70]. Furthermore, the diminished anti-tumour response of those CAR-modified NK cells was connected to increased expression levels of immunosuppressive human leukocyte antigen G (HLA-G) on ES cells, thereby mediating tumour immune escape [71].…”

Engineering Natural Killer Cells for Cancer Immunotherapy

“…In vitro, NK cells exert cytotoxicity against a number of hematologic malignancies, including acute myeloid leukemia (AML), 26-28 ALL, 29-33 multiple myeloma (MM), [34][35][36][37] as well as many solid tumors including neuroblastoma, ovarian, colon, renal cell, and gastric carcinomas. 33,[38][39][40][41][42] However, many tumors develop evasion strategies to circumvent killing by autologous NK cells. 27,31 For example, some leukemia and lymphoma cells maintain high surface expression of HLA molecules, making them invisible to NK attack, 31,32 or may lack ligands that signal through activating NK cell receptors.…”

Challenges of NK cell-based immunotherapy in the new era

“…Overall, standardized measurements for the production and quality control of NK cells are lacking, possibly causing substantial variations in therapeutic outcomes. [34]. CAR-NK cells targeting well-known targets, such as CD19, CD20, SLAMP7, and EpCAM, have been tested in vivo with in-mouse models or in vitro.…”